Human blood pressure and stress/immune responses are controlled by the steroid hormones aldosterone and cortisol, respectively. There are multiple disease states in which these hormones are overproduced and for which selective inhibitors would be clinically beneficial. However, the development of selective inhibitors of aldosterone production by cytochrome P450 11B2 (CYP11B2) and cortisol production by cytochrome P450 11B1 (CYP11B1) has been frustrated by their 93% amino acid sequence identity. The current proposal seeks to rectify this gap in knowledge by determining structural features of CYP11B1 that underlie functional differences from CYP11B2. The central hypothesis is that structural and functional studies will reveal unique CYP11B1 characteristics that could be harnessed to support the design of isoform-selective drugs. To probe differences in CYP11B enzyme interactions with ligands, we will determine Xray structures of CYP11B1 with its substrate and several inhibitors that currently under development but are non-optimal because of poor selectivity. Preliminary data suggests structural differences compared to the existing corresponding CYP11B2 structure, distinctions that might be exploited clinically (aim 1). Preliminary data also suggests that the shared required redox partner adrenodoxin interacts differently with the two CYP11B enzymes, which may provide an orthogonal approach to selective inhibition?one that doesn?t focus on the active site. Thus an X-ray structure will also be determined for CYP11B1 interacting with adrenodoxin by employing a fusion protein approach (aim 2). The final aim probes different conformations of CYP11B1 that are likely to exist in solution and in the membrane where this protein is normally embedded. All membrane P450 structures have been determined by X-ray crystallography and packing interactions occur via the membrane-binding surface through which ligands enter/exit. Thus, while these proteins are known to be flexible and dynamic, X-ray crystallography often only reveals one form, which may not be the most appropriate conformation for inhibitor design. Application of cryo-EM to CYP11B1 (aim 3) is an orthogonal approach to define more relevant conformations of this enzyme. Thus, the proposed research will determine the structures and define new interactions controlling the activity of CYP11B1. Contrasting this new information with CYP11B2 can subsequently be used to facilitate the development of clinically-relevant molecules inhibitors specific for each enzyme.